A randomized, double‐blind, controlled study evaluating the effects of two facial serums on skin aging

Abstract Background Skin aging is a natural process that occurs because of oxidative stress. Facial skin aging is often concerning for individuals due to the exposure of the face. Objectives To assess and compare the effects of two anti‐aging facial serums on the following characteristics associated with facial skin aging: fine lines/wrinkles, age spots, firmness, elasticity, texture, radiance, tone, lifting, clarity, and complexion. Methods A 24‐week, double‐blind controlled study was conducted on 130 participants who were randomized into two groups: facial serum with Liposomal Blend and facial serum without Liposomal Blend. Clinical evaluations (Visual Analog Scale) and instrumental evaluations (Cutometer, SIAscope, and Clarity Pro image analysis) were performed at weeks 0 (baseline), 2, 4, 8, 12, and 24 to assess for changes in skin aging characteristics. Results A total of 123 participants completed the study; participants that used the facial serum with Liposomal Blend had significantly greater improvements in skin aging characteristics compared to those that used the facial serum without Liposomal Blend. This study shows that Liposomal Blend is a vehicle with the ability to enhance the anti‐aging properties of the ingredients within the facial serum by facilitating its delivery into the underlying layers of the skin. Higher concentration of ingredients at the site of action could potentially lead to greater damage repair and improvements in signs of facial skin aging. Conclusion By using Liposomal Blend, practitioners and pharmacists could potentially improve the delivery of the ingredients within their formulations into the skin, which may lead to increased treatment efficacy.

as Interleukins (IL)−1, IL-6, and Tumor Necrosis Factor-alpha (TNF-α), which can increase the activity of Matrix Metalloproteinases (MMPs), enzymes that degrade extracellular matrix within the dermis. 2Extracellular matrix provides structural support to the skin and is composed of collagen, a protein that contributes mass and tensile strength, and elastin, a protein with elastic characteristics. 1 The decline in cell count and increased extracellular matrix degradation therefore causes alterations in the structure and function of the skin layers, resulting in skin aging. 2 The process of skin aging has an effect on the whole body.However, facial skin aging is often more concerning for individuals as the face is a part of the body that is visible to others. 3Characteristics associated with facial skin aging include: fine lines/wrinkles; alterations in skin firmness, elasticity, texture, and lifting; development of age spots; and alternations in skin tone, clarity, radiance, and complexion.
Fine lines and wrinkles are folds in the skin caused by cell death and loss of extracellular matrix that provide support and structure to the skin. 4Skin firmness refers to the ability of the skin to resist change, 5 while elasticity relates to the ability of the skin to return to its original shape upon deformation. 2Reduced skin firmness and elasticity are commonly observed in aging individuals as a result of the reduction in collagen and elastin content within their skin. 2,5Texture, which refers to the degree of roughness on the skin surface, is affected by organization of the collagen fibers.A change in skin texture occurs with age because of collagen fibers reorganization, causing an increase in skin roughness. 6Lifting is a characteristic affected by thinning of the skin layers and loss of subcutaneous fat in regions below the dermis, resulting in sagging of the skin. 1 Age spots are dark regions on the skin commonly found in sun-exposed areas such as the face.As the skin ages, cell death hinders the body's ability to remove the pigments melanin and lipofuscin, resulting in pigments accumulation and the formation of age spots. 7The presence of these dark regions on the skin can affect an individual's skin tone and clarity.Tone is determined based on the pigmentation pattern of the skin, 8 while clarity refers to the presence/absence of skin markings such as blemishes. 9ose with even tone and clear skin will have even distribution of pigments throughout the face, with no markings.Radiance is a term used to describe the level of skin brightness, while complexion is a general term that describes the overall appearance of the skin.An individual's complexion depends on a combination of factors such as skin texture, tone, radiance, and clarity. 9ough genetics play a major role in facial skin aging, environmental factors such as ultraviolet radiation from sunlight exposure, smoking, stress, poor diet, and alcohol can accelerate the aging process and worsen facial skin aging characteristics. 10When these characteristics become too prominent, individuals often search for cosmetic interventions in an attempt to diminish signs of skin aging. 3The objective of this study is to assess and compare the effects of two anti-aging facial serums, one with Liposomal Blend, and one without Liposomal Blend, on the following characteristics associated with facial skin aging: fine lines/wrinkles, age spots, firmness, elasticity, texture, radiance, tone, lifting, clarity, and complexion.Liposomal Blend is a permeation enhancer that is used topically as a vehicle in cosmetics and pharmaceutical formulations.It consists of proprietary liposomal components designed to enhance the delivery of substances into the underlying layers of the skin.It includes 0.05% to 5% of one or more phospholipids; 1% to 20% of Pracaxi oil that is used as a permeation enhancer in the natural composition and that decreases irritation and provides hydration and restores a cutaneous permeability of skin; 0.1% to 3% of one or more skin lipids; 1% to 10% of a butter having linoleic acid and linolenic acid; and 10% to 90% of purified water, wherein a substantial portion of liposomes of the natural liposomal composition are about 5 to 20 microns. 11The liposomal constituents have potentials to attach to skin surfaces and fuse with the lipid bilayer, facilitating delivery of ingredients into the skin. 12,13e two facial serums were developed for the purposes of preventing and improving the appearance of aging skin, and contain the following ingredients with proposed anti-aging activities: caffeine, pracaxi oil, Plukenetia volubilis seed oil, Carthamus tinctorius seed oil, palmitoyl tripeptide-5, hexapeptide-9, acetyl-dipeptide-1 cetyl ester, niacinamide, and methylsulfonylmethane (MSM).Caffeine is a naturally occurring substance with antioxidant activity.When applied to human skin fibroblasts, cells that produce collagen, caffeine helps to reduce ROS-mediated cellular damage and necrosis. 14Pracaxi oil, extracted from the seeds of Pentaclethra macroloba tree found in the Amazon forest, contains large amounts of fatty acids that play an essential role in the construction of cell membranes within the stratum corneum and epidermis.The fatty acids from pracaxi oil also have anti-inflammatory properties and could potentially protect the skin from damage by ultraviolet radiation. 15Plukenetia volubilis seed oil, extracted from a shrub plant native to the Amazon as well, contains omega-3 and omega-6 fatty acids with antioxidant activity.Studies have shown that Plukenetia volubilis extract also has the ability to facilitate fibroblast migration. 16Carthamus tinctorius (safflower) seed oil contains fatty acids with antioxidant and anti-inflammatory activities. 17Palmitoyl tripeptide-5 is a peptide that exerts its anti-aging effects by activating Tissue Growth Factor (TGB)-β, which increases collagen synthesis within the skin. 18,19Hexapeptide-9 has the ability to facilitate skin regeneration 10 while acetyl-dipeptide-1 cetyl ester was used for its skin soothing effects. 20Niacinamide (vitamin B3) is a water soluble vitamin that is a precursor to coenzymes involved in many metabolic reactions within the skin.Results from studies conducted with topical niacinamide show that in addition to having antioxidant activities, this vitamin can help improve skin texture, as well as the appearance of wrinkles and age spots. 21,22MSM is a compound that contributes sulfur to replenish glutathione levels within the body, resulting in a reduction in ROS-mediated cellular damage as glutathione is a free radical scavenger. 23

MATERIALS AND METHODS
A 24 weeks, randomized, double-blind controlled study was conducted to assess and compare the effects of two anti-aging facial serums, one serum with Liposomal Blend, and another serum without Liposomal Blend, on characteristics associated with facial skin TA B L E 1 Inclusion and exclusion criteria for enrollment of the study participants.

Inclusion criteria Exclusion criteria
• Healthy females between 39 to 65 years of age at the time of enrollment.• Able to understand and willing to complete a brief personal history, review and sign an informed consent document, and follow all study instructions.
• Lines/wrinkles in the periorbital area with a score greater than 2 cm and less than 8 cm on a standard 10 cm visual analog scale (VAS).
• Willing to come to the observation site without makeup.
• Willing to continue use of all regular brands of color cosmetics provided they do not make any moisturizing or anti-aging claims.
• Willing to refrain from using new facial products other than the assigned test materials.
• Pregnant, breast-feeding, or planning a pregnancy during the course of the study.
• Initiated the use of topical or oral prescription steroids and/or anti-inflammatory medications within 30 days of study enrollment.
• Participating in any other clinical studies.
• Known allergies to personal care product(s) (self-reported).
• History of sensitivity to skin treatment products.
• Any dermatologic disorders that may interfere with evaluation of test product performance.
• Any condition(s) apparent at entry or recognized after entry that are likely to invalidate a subject's consent to participate in this study and/or limit the ability of a subject to regularly attend all study visits or to comply with all other protocol requirements such as: diseases, injuries, alcoholism, drug abuse, psychosis, antagonistic personality, poor motivation, infirmity disability, other problems that may be emotional, intellectual, psychological or social.
• An employee of the testing facility or other testing firms/laboratories, cosmetic or raw goods manufacturers or suppliers.
aging.This study was conducted in Port Chester, NY by International Research Services Inc., and informed consent was obtained from all study participants.

Study design
A total of 130 female participants were enrolled in this study based on the inclusion and exclusion criteria displayed in Table 1.Enrollment was followed by a washout period of 1 week in which participants were instructed to wash their face with Camay soap and warm water, at least two times a day, and to apply Purpose lotion SPF 15 daily.The use of any topical facial treatment products was prohibited during the washout period and throughout the course of this study. 24Aside from the Camay soap and Purpose lotion SPF 15, participants were allowed to continue the use of color cosmetics provided that their packaging did not make any claims regarding moisturizing and/or anti-aging.
At the end of the washout period, participants were randomized into two groups: facial serum with Liposomal Blend (group A) and facial serum without Liposomal Blend (group B), with 65 participants in each group.At the baseline visit, participants were provided the assigned facial serum and were shown how to apply the product to the entire face, including the areas under the eye.Participants were instructed to apply the serum twice daily for a period of 24 weeks.In order to record compliance, participants were given a diary to record the times that they used the product for every day of the study.It was established that participants would be disqualified if compliance was lower than 80%.
Visits to the testing facility occurred at week 0 (baseline), 2, 4, 8, and 12 for all participants, while a subgroup of participants were evaluated through to week 24.Participants were instructed to wash their face at least 1 to 2 h prior to each visit.Upon arrival, participants remained at room temperature for at least 15 min to allow the skin to equilibrate to ambient indoor conditions prior to evaluation. 24inical and instrumental evaluations were then performed to measure for changes in skin aging characteristics, from baseline, following the use of the assigned facial serum.A self-assessment questionnaire TA B L E 2 Self-assessment questionnaire.2, consists of 10 statements to which participants were instructed to answer based on whether or not they agreed with the specific statement.

Clinical evaluations
The facial skin of all participants was clinically graded by a trained evaluator using the Visual Analog Scale (VAS), a tool that can be utilized to quantify subjective characteristics, 25 such as those shown in Figure 1.VAS scales have been widely used in previous studies to examine the effects of various anti-aging formulations on facial skin aging. 24,26Evaluators were instructed to specify their level of agreement to a statement by indicating a position along a 10 cm line between the two end-points, one end being 0 and the other end being 10.Signs of photo-aging can be categorized as follows: mild (1 to 3.9), moderate (4 to 6.9), or severe (7-10). 24The same evaluator performed all assessments throughout the study.

F I G U R E 1
Visual Analog Scales (VAS) for skin aging clinical evaluations.

Cutometer
The Cutometer MPA580 (Courage and Khazaka Electronic GmbH; Köln, Germany) is an instrument that allows the characterization of the mechanical properties of the skin such as firmness and elasticity. 27,28is instrument consists of a pressure generator connected to a handheld probe with a 4 mm opening.Once the probe establishes contact with the skin surface, a constant negative pressure from the generator stretches and pulls the skin into the opening of the probe.After a specified time interval of suction, the probe then releases the skin and allows it to recover in shape. 27This suction and release procedure, which is typically repeated for at least 3 cycles, was performed on one side of the face, in the region of the cheek, at baseline and each subsequent visit.The investigators used an individual face map to identify the same facial area for measurements at each visit.Changes in skin shape that occur during the suction and release cycles are detected and recorded every 0.1 s. 29 by an optical system within the probe, which consists of an infrared light source transmitting to a receptor. 27The intensity of the light transmitted is dependent on the amount of skin that penetrates the probe during suction and release, creating a straintime curve that depicts the degree of skin deformation in millimeters (mm) over time.The curve is then used in waveform analysis by the Cutometer software to measure the amount of skin raised by the suction probe (i.e., height of the curve); the amount of force required to induce the stretch; and the rate at which the skin returns to equilibrium, generating the waveform variables R0 and R5, 28 which represent skin firmness and elasticity, respectively.

SIAscope
The skin content of melanin and collagen was quantified via spectrophotometric intracutaneous analysis using the Cosmetrics SIAscope (Astron Clinica Ltd.; Cambridgeshire, United Kingdom), an instrument that consists of a probe connected to a laptop computer.
The probe is hand-held and is first placed in direct contact with the skin surface on one side of the face.The light-emitting diodes inside of the probe then shine a spectrum of visible and infrared light onto the skin at discreet wavelengths of 400 to 1000 nm. 24,30The light establishes contact with the skin surface and is absorbed by chromophores such as melanin 31 scattered by molecules such as collagen 32 or reflected.The probe is programmed to capture the reflected light and information is relayed to the SIAscope software on the laptop computer. 30The software performs a series of calculations to generate a high resolution image that shows the melanin and collagen in the skin at each wavelength, along with chromophore maps that were retrieved up to 2 mm in depth and 11 mm in circumference.These chromophore maps were used to determine the concentration and distribution of melanin and collagen within the skin layers. 31Previous studies have shown that the method of chromophore mapping can be used to assist in the diagnosis of malignant skin conditions such as melanoma, 30,31 as well as to assess for changes in characteristics associated with skin aging. 24,33Chromophore mapping using the Cosmetrics SIAscope was performed on all participants in this study at baseline, weeks 8, 12, and 24.As before, the investigators used an individual face map to identify the same facial area for measurements at each visit.

Clarity Pro
The Clarity Pro (BrighTex Bio-Photonics; San Jose, CA) is an instrument with the ability to capture and generate a three-dimensional (3D) image of the face using multi-spectral lighting. 34The facial skin of 20 participants from each group was analyzed using the Clarity Pro imaging system at baseline, weeks 4, 8, 12, and 24.Participants were instructed to position their chin on the instrument's chin rest, with the forehead touching a stopper to ensure that there was a constant and reproducible distance between the camera and each participant's face.
Frontal and lateral (45 • ) white and blue light images of the face were then captured by three cameras with specialized lenses that operate in conjunction with one another. 35These images were further analyzed by a computer software within the instrument using mathematical algorithms that allow for quantification of skin aging characteristics, as well as visualization of the skin's condition on and beneath the facial skin surface. 34,35The Clarity Pro has been used in previous studies also as an objective method for measuring facial skin redness associated with rosacea, 35 and wrinkle characteristics associated with skin aging. 34

RESULTS
A total of 123 female participants completed the study, with 63 participants in group A and 60 participants in group B. Of the seven participants that discontinued the study, two participants experienced mild facial itching and redness, while the remainder participants withdrew for personal reasons unrelated to product use.According to the daily diary, compliance was over 90% for all the 123 participants.Demographics information of the study participants is shown on Table 3.The majority of the participants were purposely Caucasian as facial skin aging is more prominent in this race group.African Americans, Hispanics and multi-ethnic participants were also included in this study though the results obtained were grouped and analyzed altogether.Although it would have been interesting to include men in this study to observe the permeation enhancer in relationship to skin thickness, the primary goal of this study was to compare the effects of two anti-aging facial serums in female subjects, who commonly suffer changes in skin aging characteristics due to hormonal imbalances, particularly around menopause.Mean ± standard deviation (SD) of clinical and instrumental scores were calculated for each group at baseline and at each visit.Differences in scores between baseline and subsequent time points were calculated for both groups.Scores obtained from clinical evaluations and instrumental analyses were compared between groups using an un-paired t-test, with p ≤ 0.05 denoting statistical significance.Chi-squared analysis was performed to compare scores obtained from the self-assessment questionnaire.

Clinical evaluations
VAS was used to quantify change in skin aging characteristics.An increase in mean VAS scores from baseline was observed at weeks 2 and 4 visits for the majority of characteristics examined.Differences between the two groups were not statistically significant (p > 0.05) for all characteristics, with the exception of skin clarity, where mean difference in VAS scores from baseline was significantly lower for group B (−0.01 ± 0.72) in comparison to group A (0.26 ± 0.73), p = 0.042 (Figure 2).At the week 8 visit, group A had a reduction in mean VAS scores for all characteristics, while fluctuations in scores were observed for group B. Furthermore, group A's score for skin texture was significantly lower than group B at weeks 8 (p = 0.021) and 12 (p = 0.037) visits, as displayed in Figure 3. Difference in skin texture scores between the two groups continued to be significant at week 24 (p = 0.012), along with group A having significantly lower scores than group B in terms of radiance (p = 0.004), tone (p = 0.017), lifting (p = 0.014), clarity (p = 0.047), and complexion health (p = 0.020).

Cutometer
When comparing skin firmness, a reduction in mean scores from baseline was observed for both groups at each visit following the start of treatment.Mean difference in scores from baseline for group A (−0.21 ± 0.07) was significantly lower than group B (−0.16 ± 0.06) at week 24, p = 0.022.When comparing skin elasticity, mean scores for group A increased from baseline at each subsequent visit, while group B scores increased at weeks 2, 4, and 8, with no change at week 12, and a reduction in score at week 24.Significant differences were detected between the groups at week 24, with participants in group A (0.14 ± 0.35) having higher scores than those in group B (−0.07 ± 0.28), p = 0.044, as displayed in Figure 4.

TA B L E 3
Demographics information of the study participants.F I G U R E 2 Clinical evaluations for skin clarity: VAS mean scores for Groups A and B at weeks 2 to 24, in comparison to baseline.

SIAscope
The SIAscope was used to quantify melanin and collagen levels in the skin.Results show an initial reduction from baseline in melanin levels for groups A (−7.96 ± 16.61) and B (−9.76 ± 15.77) at week 8, with no significant differences between the two groups, p = 0.537.Melanin levels continued to decrease for group A (−8.87 ± 16.21) at week 12, while a slight increase in level was detected for group B (−8.32 ± 18.82), p = 0.879.Though both groups had an increase in melanin at week 24, group A levels (5.55 ± 11.97) were significantly higher than group B (−7.70 ± 13.84), p = 0.003.Collagen levels within the skin also fluctuated between weeks 8 and 12 for both groups.However, as the study progressed, the collagen levels detected within the skin of participants in group A (2.78 ± 11.17) were significantly higher than that of group B (−6.25 ± 11.11) at week 24, p = 0.017, as displayed in Figure 5.

Clarity Pro
Selected results from Clarity Pro image analysis are shown on Table 4.
Difference in mean scores between baseline and subsequent visits were calculated for groups A and B. Based on the images captured, following the use of the facial serum with Liposomal Blend for 24 weeks, the participants had a 54.46% reduction in total number of wrinkles, 22.94%, 23.07%, and 38.34% reduction in wrinkle width, length, and severity, respectively.

Self-assessment questionnaire
The perception of the study participants with regards to the facial serums improved as a function of time (product use) for both

F I G U R E 4
Cutometer analysis for elasticity in the skin: mean scores for Groups A and B at weeks 2 to 24, in comparison to baseline.
groups, as displayed in Table 5.The majority of participants (> 50%) agreed that both facial serums improved facial skin firmness and texture/smoothness at every visit through week 24.

DISCUSSION
Alterations in skin aging characteristics following the use of the two facial serums were assessed using VAS (clinical evaluations); F I G U R E 5 SIAscope analysis for collagen in the skin: mean scores for Groups A and B at weeks 8 to 24 in comparison to baseline.

Clinical evaluations
An increase in mean VAS is indicative of skin aging.Results of this study show worsening of almost all examined skin characteristics at weeks 2 and 4 for both groups as mean VAS scores were higher in comparison to baseline.Skin clarity (Figure 2) was the only characteristic by week 4 that showed a significant difference between the participants that used

Cutometer
Skin firmness and elasticity scores were calculated based on analysis of the waveforms generated following skin suction with the cutometer.
Product use reduced the facial skin discoloration (age spots) Product use reduced the appearance of periorbital fine lines/wrinkles Product use reduced the appearance of perioral fine lines/wrinkles Product use improved facial skin firmness Product use improved facial skin elasticity Product use improved facial skin texture/smoothness Product use increased facial skin radiance Product use improved facial skin tone Product use improved facial complexion health Product use improved facial skin clarity was also conducted at baseline and each subsequent visit to assess the participant's perception of product use.The questionnaire, as displayed in Table the facial serum with Liposomal Blend (group A) and without Liposomal Blend (group B).Skin texture/smoothness, on the other hand, showed significant results from week 8 towards the end of the study, with clear improvements by group A in comparison to group B, as shown in Figure3.By week 24, the participants that used the facial serum with Liposomal Blend showed significant improvements of the following skin characteristics: texture/smoothness, radiance/luminosity, skin tone, lifting, complexion health, and clarity.The only skin characteristics that did not show statistically significant results by the clinical evaluations were wrinkles, age spots, and elasticity/firmness.These characteristics were reassessed by instrumental analysis, as discussed below.
Clarity Pro analysis for total wrinkles, average wrinkle length and width, and fine lines: Mean scores for Groups A and B at weeks 4 to 24, in comparison to baseline (p-value).
F I G U R E 3 Clinical evaluations for skin texture/smoothness: VAS mean scores for Groups A and B at weeks 2 to 24, in comparison to baseline. 28 Percentage of affirmative responses with regards to product use (self-assessment questionnaire).
TA B L E 5Bold p-values denote statistically significant results.